CURABLE COMPOSITION

Description

The present invention relates to a composition comprising a curable resin having ethylenically unsaturated polymerizable groups, an ethylenically unsaturated polymerizable monomer, at least one of an organic peroxide and hydrogen peroxide and an oxidoreductase, a kit of parts for preparing the composition and a process for forming a three-dimensional shaped part.

Thermoset resins such as unsaturated polyester resins, epoxyacrylate resins, urethane acrylate resins and the like including unsaturated polyesters, are commonly employed in a wide variety of products, such as casting materials and fiber reinforced materials. Unsaturated polyester resins are usually condensation products of dicarboxylic acids or anhydrides with difunctional alcohols, to provide backbone unsaturation needed for crosslinking. Polyester resins are usually diluted in a vinyl functional monomer such as styrene. The vinyl functional monomers are used to reduce the viscosity of the polyester resin and to act as a crosslinking agents. Polymerization is initiated by free radicals generated from ionizing radiation or by the photolytic or thermal decomposition of a radical initiator.

The radical initiator, usually a peroxide, decomposes into highly reactive peroxide radicals that start the radical polymerisation of the unsaturated polyesters with the vinyl functional monomers. The radical initiator is activated by accelerators, promoting the decomposition of the radical initiator, which eventually leads to the curing of the resin, generally at room temperature. Commonly used as accelerators are cobalt metal salts, like cobalt naphthenate, cobalt octoate, or cobalt neodecanoate. However, cobalt has an adverse impact on the environment, due to its hazardousness to humans, animal beings and plants. It cannot be readily degraded to reduce its detrimental influence and is labelled as carcinogenic. Therefore, there is a need for alternative accelerators used in the curing of unsaturated polyester resins. It has been found that certain enzymes can act as biobased accelerators for the crosslinking of certain kinds of polymers.

For example, U.S. Pat. No. 6,306,991 relates to a method of crosslinking polymers and more particularly to catalytic crosslinking of polymers having oxidatively crosslinkable functional groups. The oxidatively crosslinkable functional groups are crosslinked by contacting the oxidative polymer with a catalytic amount of an oxidizing enzyme. Nonetheless, the drawback of said method is that it is only applicable in aqueous coating compositions.

Accelerators are therefore required which are non-carcinogenic and biodegradable and may be employed in non-aqueous thermosetting resins. The present invention addresses these needs. It has been surprisingly found that a composition comprising

• • a) a curable resin or prepolymer component having ethylenically unsaturated polymerizable groups,
  • b) an ethylenically unsaturated polymerizable monomer,
  • c) an oxidoreductase and,
  • d) at least one of an organic peroxide and hydrogen peroxide,
  • wherein the composition comprises between 0.0 and 20.0% by weight of water, calculated on the total weight of the composition overcomes the drawbacks of the state of the art mentioned above.

The composition comprises a curable resin or prepolymer component having ethylenically unsaturated polymerizable groups a) and an ethylenically unsaturated polymerizable monomer b).

The term prepolymer refers to a monomer or system of monomers that have been reacted to an intermediate molecular mass state. This material is capable of further polymerization by reactive groups to a fully cured high molecular weight state. As such, mixtures of reactive polymers with un-reacted monomers may also be referred to as prepolymers.

Curing is a chemical process that produces the toughening or hardening of a polymer material by cross-linking of polymer chains. The curing process can be conducted by any method known in the art. Curing can be performed at room temperature or at elevated temperatures. It is possible to start at ambient temperature and then use the exothermal behavior of the system to achieve the temperature increase. It is also possible to force temperature increase by external heating, optionally in combination with pressure.

Suitable curable resins are thermosetting resins, for example unsaturated polyester resins, epoxy(meth)acrylate resins, urethane (meth)acrylate resin, unsaturated polyester (meth)acrylate and the like. Epoxy(meth)acrylate resins are often referred to as “vinyl ester resins”.

Typically, these unsaturated polyesters are the product of unsaturated mono- or dibasic acids with difunctional alcohols used in the manufacture of the aforementioned unsaturated polyester. Suitable examples of α,β-unsaturated dibasic acids are maleic acid, maleic anhydride, fumaric acid, itaconic acid and itaconic anhydride.

Suitable dibasic acids are phthalic acid, phthalic anhydride, halogenated phthalic anhydride, isophthalic acid, terephthalic acid, tetrahydrophthalic acid, tetrahydrophthalic anhydride, hexahydrophthalic acid, hexahydrophthalic anhydride, hexahydroterephthalic acid, hexahydroisophthalic acid, succinic acid, malonic acid, glutaric acid, adipic acid, sebacic acid, 1,12-dodecane dicarboxylic acid, 2,6-naphthalene dicarboxylic acid, 2,7-naphthalene dicarboxylic acid, 2,3-naphthalene dicarboxylic acid, 2,3-naphthalene dicarboxylic acid anhydride, 4,4′-biphenyldicarboxylic acid, as well as dialkylesters of the aforementioned, and the like.

Suitable monobasic acids include benzoic acid, stearic acid, oleic acid, linolenic acid, linoleic acid, palmitic acid and the like, including combinations thereof.

Examples for suitable multifunctional alcohols are polyhydric alcohols which include, for example, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, 2-methyl-1,3-propane diol, 1,3-butane diol, neopentylglycol, bisphenol A hydroxide, 1,4-butane diol, adduct of bisphenol A and propylene oxide or ethylene oxide, 1,2,3,4-tetrahydroxybutane, glycerin, trimethylol propane, 1,3-propane diol, 1,2-cyclohexane glycol, 1,3-cyclohexane glycol, 1,4-cyclohexane glycol, 1,4-cyclohexane dimethanol, paraxylene glycol, dicyclohexyl-4,4′-diol, 2,6-decalin glycol, 2,7-decalin glycol, and mixtures thereof. Monohydric alcohols include benzyl alcohol, hydroxy(dicyclopentadiene), cyclohexyl alcohol, 2-ethylhexyl alcohol, lauryl alcohol, stearyl alcohol, and mixtures thereof.

The urethane (meth)acrylate component is a product of a difunctional or polyfunctional isocyanate with a hydroxyl-functionalized (meth)acrylate. The preparation of urethane (meth)acrylates is well known to those skilled in the art. Suitable isocyantes may include 2,4-toluene diisocyanate, 2,6-toluene diisocyanate (TDI), 4,4′-diphenylmethane diisocyanate (MDI), 1,5-naphthalene diisocyanate (NDI), tetramethylxylene diisocyanate (TMXDI), hexamethylene diisocyanate (HDI), 4,4′-dicylohexylmethane diisocyanate (H₁₂MDI), 4,6′-xylene diisocyanate (XDI), isophorone isocyanate (IPDI), 2,2,4-trimethyl-1,6-hexamethylene diisocyanate (TMDI), paraphylene diisocyanate (PPDI), cyclohexyldiisocyanate (CHDI), 3,3′-tolidene 4,4′-diisocyanate (TODI) and 3,3′-dimethyl-diphenylmethane 4,4′-diisocyanate (DDI), including their polymeric forms.

Hydroxy-functionalized (meth)acrylates that may be used in the preparation of the urethane (meth)acrylate monomer component include hydroxyethyl methacrylate (HEM A), hydroxypropyl methacrylate (HPMA), hydroxyethyl acrylate (HEA), hydroxypropyl acrylate (HP A) and related compounds.

The epoxy(meth)acrylate may be a di(meth)acrylate of, for example, a bisphenol-type epoxy resin, novolak-type epoxy resin, 1,6-naphthalene-type epoxy resin or the like, which is obtained by means of reacting any of the aforementioned epoxy resin (alone or in combination), and a unsaturated monobasic acid under the presence of an esterification catalyst.

Concerning the ethylenically unsaturated polymerizable monomer b), it is possible to use any ethylenically unsaturated monomer and ethylenically unsaturated oligomer conventionally used in unsaturated polyester resins, which can crosslink with an unsaturated polyester. The ethylenically unsaturated polymerizacle monomer is preferably a monomer containing a vinyl group. Preferably, one of a (meth)acrylate group, styryl group, allyl group and vinylether group.

Examples of the aforementioned vinyl monomer include alpha-methylstyrene, chlorostyrene, dichlorostyrene, divinylbenzene, t-butylstyrene, vinyltoluene, vinyl acetate, diallylphthalate, triallylcyanurate, acrylic esters, (meth)acrylic esters, methyl (meth) acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, i-butyl (meth) acrylate, t-butyl (meth)acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth)acrylate, cyclohexyl (meth)acrylate, benzyl (meth) acrylate, stearyl (meth)acrylate, tridecyl (meth)acrylate, dicyclopentenyloxyethyl (meth) acrylate, ethylene glycol monomethylether (meth)acrylate, ethylene glycol monoethylether (meth)acrylate, ethylene glycol monobutylether (meth) acrylate, ethylene glycol monohexylether (meth)acrylate, ethylene glycol mono-2-ethylhexylether (meth)acrylate, propylene glycol monomethylether (meth)acrylate, propylene glycol monoethylether, propylene glycol monobutylether (meth)acrylate, (meth) acrylate, propylene glycol monohexylether (meth)acrylate, propylene glycol mono-2-ethylhexylether (meth)acrylate, ethylene glycol di(meth) acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth) acrylate, neopentyl glycol di(meth)acrylate, trimethylolpropane tri(meth) acrylate, pentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth) acrylate, dipentaerythritol hexa(meth)acrylate, N-vinylpyrolidone and the like. These aforementioned monomers may be used alone or in combination.

The enzyme used in the composition is an oxidoreductase c). Oxidoreductases are classified as EC 1. The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze published by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Every enzyme code consists of the letters “EC” followed by certain numbers separated by periods. Those numbers represent a progressively finer classification of the enzyme.

The at least one oxidoreductase is preferably a peroxidase (EC 1.11.1), which are enzymes defined as oxidoreductases acting on peroxide as electron acceptor.

More preferably, the enzyme is one or more of a laccase, polyphenol peroxidase, horseradish peroxidase, soybean peroxidase, pea peroxidase, guar beans peroxidase, garbanzo beans peroxidase, runner beans peroxidase, rice peroxidase, cotton peroxidase and mixtures thereof. Even more preferably, the enzyme is a peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6) or mixtures thereof. Most preferably, the enzyme is a horseradish peroxidase.

It is preferred, that amounts of the enzyme in the range of 0.01 to 40.00 mg/g are employed; more preferably 0.01 to 30.00 mg/g and most preferably 0.01 to 25.00 mg/g based on the weight of components a)+b). In a preferred embodiment, 0.10 to 30.00 mg/g enzyme are employed. In another preferred embodiment, 0.50 to 30.00 mg/g enzyme are employed. In a different preferred embodiment, 0.30 to 40.00 mg/g enzyme are employed, more preferred 0.30 to 30.00 mg/g and most preferred 0.30 to 25.00 mg/g enzyme, all calculated on the weight of components a)+b).

The enzymes utilized according to the invention can generally be of any origin. The oxidoreductase may suitably be from plant, fungal, mammalian, yeast or bacterial origin. Horseradish peroxidase for example is a glycoprotein with a molecular weight of about 40,000, and may suitably extracted from the horseradish root, which is a perennial plant of the family Brassicaceae. Catalase, with a molecular weight of about 240 kDa may preferably be extracted from bovine liver.

Examples of suitable fungi are Collybia, Coprinus, Fornus, Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora, Phlebia, in particular P. radiata, Coriolus, in particular C. hirsitus, Botrytis, Polyporus, in particular P. pinsitus and P. versicolor, Rhizoctonia solani, Scytalidium, in particular S. thermophilium, Pyricularia, in particular P. oryzae, Coprinus, in particular C. cinereus, Trametes, in particular T. hirsuta, T. villosa and T. versicolor, Coriolopsis gallica, Phanerochaete chrysosporium, Heterobasidion annosum, Spiniger meineckellus and Myceliophthora thermophila.

The oxidoreductase may preferably be derived from a bacterium that is selected from the group comprised of Bacillus, Pseudomonas, Streptomyces, Corynebacterium, Micrococcus and Azospirillum.

The oxidoreductase can be either directly isolated from the corresponding source or be produced by means of a recombinant technology. The oxidoreductase is suitably an extract from a natural source or produced via homologous or heterologous expression.

Heterologous expression denotes for the expression of a gene in a system that naturally do not contain the gene, e.g. certain type of bacteria. The recombinant production takes place in a different host cell thereby that the enzyme encoding DNA section is isolated from the source of origin and introduced into a host cell. Homologous expression on the other hand refers to over-expression of a gene in a system where the gene naturally exists. In both cases, the expression and over-expression of the specific gene leads to the production of the designated enzyme eventually. On the contrary, natural source denotes for the source of the enzyme, where the designated enzyme is synthesized without manipulating the gene expression by processes other than breeding and crossing. For example, the oxidoreductase may suitably be extracted from the horseradish root as natural source. Appropriate methods are sufficiently familiar to the person skilled in the art.

The composition further comprises at least one of an organic peroxide and hydrogen peroxide d). Suitable examples for organic peroxides are benzoyl peroxide, t-butyl hydroperoxide, diacylperoxides, hydroperoxides, ketone peroxides, peroxyesters, peroxyketals, dialkyl peroxides, alkyl peresters and percarbonates, methyl ethyl ketone peroxide, benzoyl peroxide, acetyl acetone peroxide and cumene hydroperoxide. Combinations of two or more peroxides may be also used to cure the resin.

Generally, the at least one of an organic peroxide and hydrogen peroxide is employed in an amount from 0.05 to 10.00% by weight, preferably 0.10 to 7.00% by weight and more preferably 0.10 to 4.00% by weight based on the total weight of the total composition.

The composition comprises 0.0 to 20.0% by weight of water. Suitably, the composition comprises 0.0 to 15.0% of water, more suitably 0.0 to 10.0% by weight of water. Most suitably, the composition comprises 0.0 to 5.0% by weight of water, calculated on the total weight of the composition.

Preferably, the composition further comprises a mediator for enzymatic initiation of radical polymerization. Examples for such mediators are 4-hydroxybenzoic acid, 4-hydroxyacetophenone, pentane-2,4-dione, nitroso compounds and hydroxyamine compounds, such as cycloaliphatic NO or NOH containing compounds, heterocyclic NO or NOH containing compounds, aromatic NO or NOH containing compounds, phenolic compounds with at least one, preferably two or more, phenolic hydroxyl group or groups, phenothiazine, phenyl compounds, heterocyclic compounds, polyoxometalates, 2,4-pentanedione and derivatives of these compounds, as well as 6-Hydroxy-2-naphthoic acid, 7-Methoxy-2-naphthol, 4-Hydroxycoumarin, n-Hydroxyphthalimide, Tetronic acid, 1,3-Cyclopentanedione, 10-Methylphenothiazine, Phenylacetic acid, 4-Hydroxybenzonitrile, 4-Hydroxybenzyl alcohol, 4-Hydroxybenzaldehyde, 4-Hydroxybenzoic acid, 4″-Hydroxyacetanilide, 4″-Hydroxyacetophenone, 4″-hydroxy-4-biphenylcarboxylic acid, 4″-hydroxy-4-biphenylcarboxylic acid, Vanillin, Dibenzoylmethane, Benzoylacetone and mixtures thereof. It is preferred that the mediator is at least one of 6-hydroxy-2-naphtoic acid or Acetylacetone (ACAC).

The mediator is preferably present in an amount of 0.01 to 5.00 mmol/g, more preferably 0.02 to 3.00 mmol/g and most preferably 0.02 to 2.00 mmol/g, based on the weight of components a)+b).

Suitably, the composition is liquid at a temperature of 23° C.

The composition generally comprises between 0.0 and 40.0% of organic solvent. An example of a suitable organic solvent is dimethyl sulfoxide. Preferably, the organic solvent is present in an amount of 0.5 to 30.0% by weight, more preferably in an amount of 1.0 to 20.0% by weight and most preferably 1.0 to 10.0% by weight, calculated on the total weight of the composition.

Moreover, the composition suitably comprises solid particles selected from fillers, pigments, fibers, and combinations thereof. Fillers may be used at levels up to about 60% by weight, based on the total weight of the composition and may include calcium carbonate, calcium sulfate, aluminum, aluminium trihydrate, aluminium hydroxide, hydraulic silicates, clay, talc, barium sulfate, silica powder, glass powder, glass beads, microcellulose, silica sand, river sand, marble waste, crushed stone or any combinations thereof.

Fiber reinforced composite materials comprise fibers embedded in a polymer matrix. The polymer matrix serves as binder between the fibers. The fibers generally improve the mechanical properties of composite material, as compared to the matrix polymer alone. The fibers may be inorganic or organic. Suitable fibers are glass fiber, carbon fiber, basalt fiber and polymeric fibers. They may generally be employed up to 60% by weight, based on the total weight of the composition.

Carbon fibers include amorphous carbon fibers and graphite fibers. Carbon fibers produced from various starting materials are equally suitable, for example, carbon fibers prepared from polyacrylonitrile, pitch, or rayon. The carbon fibers may have undergone a chemical or mechanical surface pretreatment, for example with known sizing agents during fiber manufacture. Carbon fibers, which have not been subjected to specific pretreatments, may likewise be employed. Depending on the intended end use, the carbon fibers may be present as filament fibers, as staple fibers, or as chopped fibers. In some embodiments, the carbon fibers are present as a woven or non-woven fabric. On other embodiments, the carbon fibers are present as a roving.

All known kinds of pigments may be used in the composition according to the invention. Inorganic or organic pigments and mixtures thereof may suitably be employed. Typically, the organic pigments are color pigments. This refers to colored material made of organic compounds with pigment properties. In some embodiments, the inorganic and organic pigments may preferably be used up to 60% by weight, based on the total weight of the composition.

In one embodiment, the composition comprises

• • a) 30.00 to 90.00% by weight of a curable resin or prepolymer component having ethylenically unsaturated polymerizable groups,
  • b) 10.00 to 70.00% by weight of an ethylenically unsaturated polymerizable monomer,
  • c) 0.01 to 40.00 mg/g of an oxidoreductase and
  • d) 0.05 to 10.00% by weight of an at least one of an organic peroxide and hydrogen peroxide
  • wherein the composition comprises between 0.00 and 20.00% by weight of water, calculated on the total weight of the composition.

Curing of a thermosetting resin will initiate when at least all four components, i.e. the curable resin or prepolymer, the ethylenically unsaturated polymerizable monomer, the oxidoreductase and the peroxide, are mixed together. Therefore, to ensure a storage-stable composition, which can be prepared shortly before the application the invention further relates to a kit of parts for preparing the composition according to the invention, comprising

• • I. a binder module comprising
    • a) a curable resin or prepolymer component having ethylenically unsaturated polymerizable groups,
    • b) an ethylenically unsaturated polymerizable monomer, and
    • c) an oxidoreductase, and
  • II. a hardener module comprising at least one of an organic peroxide and hydrogen peroxide,
    • wherein each module comprises between 0.0 and 20.0% by weight of water, calculated on the weight of the respective module.

Additionally, the thermosetting resin can be prepared by mixing three different modules, to achieve the desired curing. Therefore, the invention also relates to a kit of parts for preparing the composition according to the invention, comprising

• • I. a binder module comprising
    • a) a curable resin or prepolymer component having ethylenically unsaturated polymerizable groups,
    • b) an ethylenically unsaturated polymerizable monomer,
  • II. a hardener module comprising at least one of an organic peroxide and hydrogen peroxide, and
  • III. an activator module comprising an oxidoreductase,
    • wherein the binder module and the hardener module comprise between 0.0 and 20.0% by weight of water, calculated on the weight of the respective module.

The invention also deals with a process of forming a three-dimensional shaped part comprising the steps of

• • i. preparing a composition according to the invention,
  • ii. bringing the composition in a desired three-dimensional shape, and
  • iii. curing the composition by radical polymerization.

Examples for three-dimensional shaped parts are components or parts of boats, tanks (e.g. for oil, water, chemical products), pipes and tubes (e.g. for drinking and waste water), wall covering and sheathing, caravans, bathroom and lavatory interior (e.g. sinks, bathtubs, shower trays), seats (e.g. for busses, trains, stadiums), parts for automotives like cars, trucks, tractors (e.g. radiator cowls, trunk lids, air deflectors, spoilers, attachment parts, roofs), doors, window and casement frames, profiles, battery housings, parts for wind power plants like blades, housings, and the like.

Step ii) of the process of forming a three-dimensional shaped part is preferably carried out by introducing the composition according to the invention in a mold.

The process of forming a three-dimensional shaped part may suitably include the step of impregnating fibers with the composition according to the invention. Examples for preferred fibers are mentioned above.

Typical procedures comprise sheet molding compounding (SMC), bulk molding compounding (BMC), infusion molding (RIM—resin infusion molding, RTM—resin transfer molding), compression molding, VARI (vacuum applied resin infusion), filament winding, pultrusion, and autoclave curing.

Further components may be present in the composition, in particular such components which are typically used in manufacturing thermosetting resins. Examples of such components include thickeners, UV stabilizers, mold release agents, wetting and dispersing additives, rheology additives, surface additives and anti-foaming agents.

EXAMPLES

Description of Raw Materials Used

TABLE 1
Raw materials

Trade
designation	Chemical description	Supplier
Palatal P4-01	UP resin based on orthophthalic acid	AOC
	and standard glycols dissolved in	Aliancys
	>25-<50% styrene
Derakane	Epoxy vinyl ester resin based on	Ashland
Momentum 411-	bisphenol-A epoxy resin dissolved in
350	>40-<50% styrene
Palatal A-410	UP resin based on isophthalic acid	AOC
	and neopentylglycol dissolved in	Aliancys
	>25-<50% styrene
Palatal A 400-01	UP resin based on isophthalic acid	AOC
FC	and standard glycols dissolved in	Aliancys
	>25-<50% styrene
Advalite VH-1223	Vinyl hybrid resin, styrene free	Reichhold
Advalite Vinyl	Vinyl hybrid resin	Reichhold
hybrid 35060-00
Atlac Premium	Vinyl ester resin dissolved in	AOC
100	methacrylate, styrene free	Aliancys
Butanox M-50	Methyl ethyl ketone peroxide	Akzo Nobel
	dissolved in dimethylphthalate
Trigonox 44B	Acetylacetone peroxide dissolved in	Akzo Nobel
	solvent mixture
Butanox LPT-IN	Methyl ethyl ketone peroxide	Akzo Nobel
	dissolved in diisononylphthalate
Butanox M-50VR	Methyl ethyl ketone peroxide	Akzo Nobel
	dissolved in dimethylphthalate with
	dye
	Hydrogen peroxide, 30%	Merck
	Dimethyl sulfoxide, Ph. Eur.	VWR
	Water, ultrapure
Rhodiasolve	Pentanoic acid, 5-(dimethylamino)-2-	Solvay
PolarClean HSP	methyl-5-oxo-methyl ester
Tamisolve NxG	1-Butylpyrrolidin-2-one	Eastman
	2-Pyrrolidone
	n-Formylmorpholine
	n-Methyl-2-pyrrolidone
	Peroxidase	various
		supplier
	Catalase	various
		supplier
	Acetylacetone (ACAC)	Sigma
		Aldrich
	6-Hydroxy-2-naphtoic acid	AlfaAesar
NL-49P	Cobalt(ll) 2-ethylhexanoate, 1% Co	Akzo Nobel

Sample Preparation

10 g resin (Palatal P4-01 if not mentioned otherwise) were placed in a 50 ml PE-plastic beaker. Mediators in solid form (like 6-Hydroxy-2-naphtoic acid) were dissolved in DMSO or water. Liquid mediators (like ACAC) were added directly to resin. The solid enzyme was placed in a 50 ml centrifugation tube or 5 ml reaction tube, depending on needed final amount, and was dissolved in mediator-DMSO-solution, water, or pure DMSO. Liquid enzyme was added directly to the resin. The enzyme solution was vortexed and shortly centrifuged at 4° C. and 6000 rcf. All components were added to the resin as follows: first the enzyme-(mediator)-solution and the peroxide at the end. In case of the mediator being liquid, the enzyme-solution was added first and the mediator was added afterwards, followed by addition of the peroxide. After each addition, the mixture was stirred with a spatula by hand. All w % are calculated on the weight of the resin (component a+b).

Shore Hardness Measurement

After addition of all components, plastic beakers were closed with a cap. Incubation was performed in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid. Shore A hardness measurements of samples were made with digitest II Type DTAA (Bareiss) after 2, 24 and 48 hours according to DIN ISO 7619.

Determination of Gel-Time

After addition of all components, samples were directly filled in test tubes (Duran #261302106) up to the mark (4 cm), corresponding to about 5 g of sample. Measurement was started immediately and gel-time was measured up to 2 hours at 23° C. with Gelnorm-Geltimer (Gel Instrumente AG) with measurement pin (H.Saur #020.30).

Non-Inventive Control with Accelerator NL-49P and Addition of Water

The samples were prepared as described above (see sample preparation) using the amounts as described in table 2 followed by incubation in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid.

	TABLE 2
	water:	0-10.0	wt %
	accelerator (NL-49P):	1	wt %
	peroxide (Butanox M-50):	2	wt %

TABLE 3
Results

	water	started to
	[wt %]	cure [h]

	0	<0.15
	4.3	15
	6.0	15
	8.0	24
	10.0	24

Started to cure was determined by poking the reaction mixture with a spatula. When poking with a spatula left an indentation in the reaction mixture, and no flow-back of the reaction mixture to the indentation was observed, gelation was considered to have occurred.

The table shows that an increased addition of water resulted in an increased reaction time of curing with the Cobalt accelerator.

Variation of Enzyme Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 4 followed by incubation in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in 4.3 wt % water.

	TABLE 4
	enzyme peroxidase, solid:	0-17 mg/g resin
	water:	4.3 wt %
	mediator (ACAC):	0.12 mmol/g resin
	peroxide (Butanox M50):	2 wt %

TABLE 5
Results

enzyme

started to cure [h]

[mg solid/g	with	without
resin]	mediator	mediator

0.0	x	x
0.4	x	167
2.0	65	65
3.0	49	49
4.0	42	42
7.0	24	49
9.0	18	17
13.0	15	15
17.0	7	15
x: “no curing”

The curing was observed visually and determined as described above.

The results show that with an increasing amount of enzyme the reaction time decreased. By addition of the mediator, the reaction time decreased further.

Variation of Water Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 6 followed by incubation in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in various water concentrations.

	TABLE 6
	enzyme peroxidase, solid:	17 mg/g resin
	water:	4-10 wt %
	mediator (ACAC):	1.2 mmol/g resin
	peroxide (Butanox M-50):	2 wt %

TABLE 7
Results

started to cure [min]

water [wt %]	with mediator	without mediator

4	1440	1440
6	60	180
8	60	60
10	60	60

Curing was observed visually and determined as described above.

With increasing water content, the reaction time decreased. With addition of mediator, the reaction time decreased even further.

Variation of Peroxide Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 8 followed by incubation in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in 10 wt % water.

	TABLE 8
	enzyme peroxidase, solid:	17 mg/g resin
	water:	10 wt %
	mediator (ACAC):	1.2 mmol/g resin
	peroxide (Butanox M-50):	0.2-6.0 wt %

TABLE 9
Results

started to cure [min]

peroxide [wt %]	with mediator	without mediator

0.2	60	120
1.0	36	36
2.0	36	500
4.0	300	1440
6.0	180	480

Curing was observed visually and determined as described above.

In general, lower peroxide concentrations showed shorter reaction times. The best result was achieved between 0.2 and 1.0 wt % peroxide.

Variation of Mediator Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 10 followed by incubation in a Thermomixer comfort (Eppendorf) for 2, 24, 48 hours at 24° C. with closed lid. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in 10 wt % water.

	TABLE 10
	enzyme peroxidase, solid:	17 mg/g resin
	water:	10 wt %
	mediator (ACAC):	0.00-0.24 mmol/g resin
	peroxide (Butanox M-50):	1 wt %

TABLE 11
Results

mediator

[mmol/g

started to

visual evaluation

	resin]	cure [min]	after 2 h	after 24 h	after 48 h
	0.00	0-36	3.0	2.0	1.7
	0.01	0-36	3.3	1.7	1.3
	0.06	0-36	2.3	1.3	1.3
	0.12	0-36	2.7	1.7	1.3
	0.24	0-36	3.0	2.0	1.7
	1 = very good; 2 = good; 3 = satisfactory

Curing was observed visually and determined as described above.

Moreover, the hardness, thickness and homogeneity of the resin were assessed. The parameters were evaluated visually and additionally by poking the resin with a spatula. Subsequently, the criteria summarized in the table above were applied. 3 denotes for medium thick sample, which is soft to solid with residual liquid; 2 denotes for a thick sample, solid and with a lower content of residual liquid and 1 denotes for a thick sample, which is very solid with almost no residual liquid.

The best results were achieved between 0.01 and 0.06 mmol ACAC/g resin.

Increase of Water Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 12. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in water concentrations between 10 and 20 w %.

	TABLE 12
	enzyme peroxidase, solid:	17 mg/g resin
	water:	10-20 wt %
	mediator (ACAC):	0.05 mmol/g resin
	peroxide (Butanox M-50):	0.5 wt %

TABLE 13
Results

			Shore A after	Shore A after
	water [wt %]	gel-time [min]	24 h	48 h

	10	22	71	80
	15	0	66	76
	20	50	47	78

The best result was reached with 10 wt % water content. With higher water concentrations, the gel-time increased and but the Shore A hardness of the final sample decreased.

Peroxidases from Various Suppliers

The samples were prepared as described above (see sample preparation) using the amounts as described in table 14. The enzymes were dissolved in 10 wt % water.

	TABLE 14
	enzyme peroxidase, solid:	17 mg/g resin
	water:	10 wt %
	mediator (ACAC):	0.5 mmol/g resin
	peroxide (Butanox M-50):	0.5 wt %

The activity of the enzyme was determined by using ABTS as substrate. 1 U stands for one unit which oxidizes 1.0 μmole of 2,2″-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) per minute at pH 5.0 at 25° C., measured at A=405 nm (Spark, Tecan).

TABLE 15
Results

		enzyme
		activity	gel-	Shore A	Shore A
	article	[U/mg	time	after	after
supplier	number	protein]	[min]	24 h	48 h

abcr	AB348238	313	41	44	77
Alfa Aesar	J60026	109	58	41	76
Amano	Amano PO-3	652	11	83	85
Amresco	417	n/a	20	73	76
BBI	161451BBI	497	25	79	85
Solutions	161453BBI	443	81	48	77
	161455BBI	412	133	52	87
	161457BBI	256	32	44	66
Biosynth	P-2000	236	92	33	71
Calzyme	100A0400	577	29	89	81
	100A0600	519	16	81	89
Chemical	CP9003-99-0-	291	15	42	76
Point	BULK
Creative	PHAM-231	177	24	69	83
Enzymes
Faizyme	16001	356	16	73	82
	16002	348	19	68	77
	16004	196	n/a	20	56
	16005	169	n/a	20	58
Iris	LS-1217	266	37	43	76
Biotech
Proactive	P113-0165	260	16	59	69
Molecular
Research
Sigma	P8125	175	51	40	70
Aldrich
	P8250	325	18	80	91
	P8375	427	14	77	84
TCI	P0073	318	17	73	80
Chemicals

From the table it is visible, that all peroxidases showed a curing effect, irrespective of their origin from different commercial suppliers.

DMSO as Solvent

The samples were prepared as described above (see sample preparation) using the amounts as described in table 16. The enzyme (peroxidase, Sigma Aldrich, #P8250) was dissolved in various concentrations of water-DMSO mixtures. No mediator was used in this experiment.

	TABLE 16
	enzyme peroxidase, solid:	17 mg/g resin
	peroxide (Butanox M-50):	0.2 wt %

TABLE 17
Results

water	DMSO	gel-time	Shore A	Shore A
[wt %]	[wt %]	[min]	after 24 h	after 48 h

0	10	49	79	82
9	1	86	78	84
5	5	58	79	83
Without enzyme, no curing was detectable.

From the table it is visible that curing of the resin with peroxidase was also possible without any water and mediator in the system.

Curing with Various Resins and Peroxides

Experiments with Cobalt accelerator NL-49P (without enzyme) were performed as non-inventive control. The samples were prepared as described above (see sample preparation) using the amounts as described in table 18.

TABLE 18
			NL-
		peroxide	49P	gel-time	Shore A	Shore A
resin	peroxide	[wt %]	[wt %]	[min]	after 24 h	after 48 h

Palatal P 4-01	Butanox M-	2	1.0	7	88	96
	50
Palatal P 4-01	Trigonox	1	0.5	16	90	93
	44B
Palatal A-410	Trigonox	1	0.5	10	90	94
	44B
Palatal A 400-	Butanox M-	1	1.0	7	91	95
01 FC	50
Atlac Premium	Butanox	2	2.0	13	85	91
100	LPT-IN

Experiments with Different Resins

The enzyme (peroxidase, BBI Solutions, #161451BBI) was dissolved in 10 wt % water.

	TABLE 19
	enzyme peroxidase, solid:	17 mg/g resin
	mediator (ACAC):	0.05 mmol/g resin
	Peroxide (Butanox M-50):	0.5 wt %

TABLE 20
Results

	gel-time	Shore A	Shore A
resin	[min]	after 24 h	after 48 h
Palatal P 4-01	19	70	82
Palatal A-410	10	75	89
Palatal A 400-01 FC	7	87	89
Derakane Momentum 411-350	18	85	94
Advalite VH-1223	39	29	33
Advalite ™ Vinyl hybrid 35060-00	n/a	24	37
Atlac Premium 100	n/a	65	77

With peroxidase, different resin systems based on unsaturated polyester, vinylester, or with acrylates or styrene as monomer could be cured.

For testing of different peroxides, Palatal P 4-01 as resin was used.

TABLE 21
Results

				Shore A
		gel-time	Shore A	after
resin	peroxide	[min]	after 24 h	48 h
Palatal P 4-01	Butanox LPT-	31	88	87
	IN
Palatal P 4-01	Butanox M-	25	64	79
	50VR
Palatal P 4-01	Butanox M-50	19	70	82

With peroxidase, different peroxides could be used for curing of the resin system.

Variation of DMSO Concentration

The samples were prepared as described above (see sample preparation) using the amounts as described in table 22. The enzyme (peroxidase, Faizyme, #16001) was dissolved in various DMSO concentrations. 6-hydroxy-2-naphtoic acid was used as mediator.

	TABLE 22
	enzyme peroxidase, solid:	17 mg/g resin
	DMSO:	2.5-20 wt %
	mediator (6-hydroxy-2-naphtoic acid):	0.05 mmol/g resin
	peroxide (Butanox M-50):	0.5 wt %

TABLE 23
Results

DMSO	gel-time	Shore A	Shore A	Shore A
[wt %]	[min]	after 2 h	after 24 h	after 48 h

2.5	36	20	83	87
5.0	4	85	97	98
7.5	5	90	97	97
10.0	5	89	97	97
15.0	4	73	94	95
20.0	6	66	90	90

The best result was reached with 7.5 wt % DMSO.

Variation of Enzyme Concentration with DMSO as Solvent

The samples were prepared as described above (see sample preparation) using the amounts as described in table 24. The enzyme (peroxidase, Faizyme, #16001) was dissolved in 7.5 wt % DMSO.

	TABLE 24
	enzyme peroxidase, solid:	0-10 mg/g resin
	DMSO:	7.5 wt %
	mediator (6-hydroxy-2-naphtoic	0.05 mmol/g
	acid):	resin
	peroxide (Butanox M-50):	0.5 wt %

TABLE 25
Results

	enzyme, solid	gel-time	Shore A	Shore A	Shore A
	[mg/g resin]	[min]	after 2 h	after 24 h	after 48 h

	0.0	n/a	x	x	x
	2.5	271	x	71	87
	5.0	30	59	91	92
	7.5	16	67	94	95
	10.0	10	77	97	96
	x: no curing was detectable

The samples were prepared as described above (see sample preparation) using the amounts as described in table 26. Enzyme (peroxidase, Faizyme, #16001) was dissolved in 7.5 wt % DMSO.

	TABLE 26
	enzyme peroxidase, solid:	0-10 mg/g resin
	DMSO:	7.5 wt %
	mediator (6-hydroxy-2-naphtoic	0.1 mmol/g resin
	acid):
	peroxide (Butanox M-50):	0.5 wt %

TABLE 27
Results

enzyme,			Shore A	Shore A
solid	geltime		after	after
[mg/g resin]	[min]	Shore A after 2 h	24 h	48 h

0.00	x	x	x	x
0.25	1387	x	x	0*
0.50	214	0	84	87
1.00	49	21	80	83
2.50	11	75	95	94
5.00	5	92	96	96
7.50	4	92	97	96
10.00	3	92	96	96
0*: Curing of sample was visible, but sample was too soft for measurement of Shore A hardness.

Also with DMSO, an increasing enzyme concentration lead to a decrease in gel-time and an increase in hardness.

Curing with H₂O₂ as Peroxide

The samples were prepared as described above (see sample preparation) using the amounts as described in table 28. The mediator 6-hydroxy-2-naphtoic acid and the enzyme (peroxidase, Faizyme, #16001) were dissolved in 10 wt % DMSO.

	TABLE 28
	enzyme peroxidase, solid:	10 mg/g resin
	water/DMSO:	10 wt %
	mediator (6-hydroxy-2-	0.05 mmol/g
	naphtoesäure/ACAC):	resin
	peroxide (H2O2, 30%):	0.1-0.3 wt %

TABLE 29
Results

	H2O2 (30%)	gel-time	Shore A	Shore A	Shore A
	[wt %]	[min]	after 2 h	after 24 h	after 48 h
	0.1	n/a	x	52	88
	0.3	n/a	x	0*	68
	0*: Curing of sample was visible, but sample was too soft for measurement of Shore A hardness.
	x: no curing was detectable

In the following, the enzyme was dissolved in 10 wt % water and ACAC was used as mediator.

TABLE 30
H2O2 (30%)	gel-time	Shore A	Shore A	Shore A after
[wt %]	[min]	after 2 h	after 24 h	48 h
0.6	n/a	x	33	79

With both solvents (DMSO or water), curing of the resin with peroxidase and H₂O₂ was possible.

Curing of Thin Layers

The samples were prepared as described above (see sample preparation) using the amounts as described in table 30. The enzyme (peroxidase, Faizyme, #16001) was dissolved in 7.5 wt % DMSO.

	TABLE 31
	enzyme peroxidase, solid:	17 mg/g resin
	mediator (6-hydroxy-2-naphtoic acid):	0.05 mmol/g resin
	peroxide (Butanox M-50)	0.5 wt %

The mixture was transferred to a glass plate and spread with a doctor blade (300 μm). The curing was tested via scratching the resin with a spatula. Additionally, a non-inventive comparison example with cobalt instead of the enzyme and mediator was tested.

Comparable curing with peroxidase or cobalt was observed. As a result, also thin film curing was possible with peroxidase.

Curing with Catalases

To test if the enzyme catalase can also be suitably used in the curing resin systems, the following experiment was carried out with catalase instead of peroxidase. The enzyme was dissolved in 10 wt % water.

	TABLE 32
	enzyme catalase, solid:	17 mg/g resin
	water:	10 wt %
	mediator (ACAC):	0.5 mmol/g resin
	peroxide (Butanox M-50):	0.5 wt %

TABLE 33
Results

		article	gel-time	Shore A	Shore A
origin	supplier	number	[min]	after 24 h	after 48 h
Bovine liver	Sigma	C40	>120	0*	0*
0*: Curing of sample was visible, but sample was too soft for measurement of Shore A hardness.

Enzyme was dissolved in 10 wt % in a mixture of DMSO and water (50:50).

	TABLE 34
	enzyme catalase, solid:	17 mg/g resin
	Dmso/water (50:50):	10 wt %
	peroxide (Butanox M-50):	0.5 wt %

TABLE 35
Results

			geltime	Shore A	Shore A	Shore A
origin	supplier	order no	[min]	after 2 h	after 24 h	after 48 h

bovine liver	Sigma	C40	n/a	x	x	0*
bovine liver	Sigma	E3289	n/a	0*	16	37
	Aldrich
0*: Curing of sample was visible, but sample was too soft for measurement of Shore A hardness.

The tables show that the employment of catalase also leads to the curing of the resin.